Altered ovarian tissue level of lysophosphatidic acid and mRNA expressions of its metabolic enzymes and receptors in rats received gonadotropin-hyperstimulation.

Lysophosphatidic acid (LPA), a well-studied member of the lysophospholipid family, is known to exert an important bio-effect on oocyte maturation and ovulation in mammals. We attempted to determine how follicle maturation in the rat ovary affects the levels of LPA and its precursor lysophospholipids, as well as mRNA levels of LPA-producing and -degrading enzymes and LPA receptors in rats that received gonadotropin-hyper-stimulation. Tissue levels of lysophospholipids were quantified by LC-MS/MS, and relative mRNA expression levels of LPA-producing and -degrading enzymes, and LPA receptors were measured by RT-PCR. Tissue levels of n-6 polyunsaturated LPAs and LPCs were higher in the ovaries of rats after receiving human chorionic gonadotropin, unlike the distinct profiles of n-3 polyunsaturated LPAs, which had lower levels, and LPCs which had higher levels, after the gonadotropin treatment. The effects of different levels of other polyunsaturated lysophospholipids were variable: decreased levels of lysophosphatidylglycerol, and unaltered levels of lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The results indicate that expression of mRNA levels of autotaxin and acylglycerol kinase were reduced and expression of lipid phosphate phosphatase 3 was elevated, whereas expressions of two membrane phosphatidic acid phosphatases (A1α and A1ß) and lipid phosphate phosphatase 1 were essentially unaltered in rat ovary at several stages after ovary hyperstimulation. After the gonadotropin treatment, the expression levels of all LPA receptors except LPA3 were decreased at various times. These results are discussed with respect to the physiological processes of the ovarian environment and development in rats.

Extracellular and intracellular productions of lysophosphatidic acids and cyclic phosphatidic acids by lysophospholipase D from exogenously added lysophosphatidylcholines to cultured NRK52E cells.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that is a notable biomarker of kidney injury. However, it is not clear how LPA is produced in renal cells. In this study, we explored LPA generation and its enzymatic pathway in a rat kidney-derived cell, NRK52E cells. Culturing of NRK52E cells with acyl lysophosphatidylcholine (acyl LPC), or lyso-platelet activating factor (lysoPAF, alkyl LPC) was resulted in increased extracellular level of choline, co-product with LPA by lysophospholipase D (lysoPLD). Their activities were enhanced by addition of calcium ions to the cell culture medium, but failed to be inhibited by S32826, an autotaxin (ATX)-specific inhibitor. Liquid chromatography-tandem mass spectrometric analysis revealed the small, but significant extracellular production of acyl LPA/cyclic phosphatidic acid (cPA) and alkyl LPA/cPA. The mRNA expression of glycerophosphodiesterase (GDE) 7 with lysoPLD activity was elevated in confluent NRK52E cells cultured over 3 days. GDE7 plasmid-transfection of NRK52E cells augmented both extracellular and intracellular productions of LPAs (acyl and alkyl) as well as extracellular productions of cPAs (acyl and alkyl) from exogenous LPCs (acyl and alkyl). These results suggest that intact NRK52E cells are able to produce choline and LPA/cPA from exogenous LPCs through the enzymatic action of GDE7 that is located on the plasma membranes and intracellular membranes.

Hypoxia increases cellular levels of phosphatidic acid and lysophospholipids in undifferentiated Caco-2 cells.

Cancer cells are known to survive in a hypoxic microenvironment by altering their lipid metabolism as well as their energy metabolism. In this study, Caco-2 cells derived from human colon cancer, were found to have elevated intracellular levels of phosphatidic acid and its lysoform, lysophosphatidic acid (LPA), under hypoxic conditions. Our results suggested that the elevation of LPA in Caco-2 cells was mainly due to the combined increases in cellular levels of lysophosphatidylcholine and lysophosphatidylethanolamine by phospholipase A2 and subsequent hydrolysis to LPA by lysophospholipase D. We detected the Ca2+ -stimulated choline-producing activities toward exogenous lysophosphatidylcholines in whole Caco-2 cell homogenates, indicating their involvement in the LPA production in intact Caco-2 cells.

Lysophosphatidic acid production from lysophosphatidylcholine by lysophospholipase D activity of autotaxin in plasma of women with normal and adverse pregnancies.

To identify biomarker lipids causing preterm delivery, we focused on lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA). The results of liquid chromatography-tandem mass spectrometry revealed that plasma levels of LPCs and LPAs were higher in the first and third (T3) trimesters of human normal and adverse pregnancies than in the second trimester, suggesting the direct metabolic conversion of LPC to LPA by lysophospholipase D (lysoPLD) activity of autotaxin. The elevated LPC and LPA levels in women with preterm deliveries in T3 were higher than in women with term deliveries under normal pregnancy in T3. We measured lysoPLD activity of diluted sera of pregnant women by quantification of choline released from exogenous LPC, and found progressive increases of lysoPLD activities in women with normal and adverse pregnancies. Ratios of lysoPLD activities for linoleoyl LPC to that for palmitoyl LPC were found to be decreased in pregnant women compared to that in non-pregnant women. These results may be due to the altered patterns of endogenous modulators for autotaxin and the profiles of the bound metal ion.

A cross-sectional study of the psychological impact of the COVID-19 pandemic on undergraduate and graduate students in Japan.

BACKGROUND: Due to the COVID-19 pandemic, a state of emergency was declared in Japan and university classes were suspended, causing concern about the deterioration of the mental health of isolated students. This study aimed to understand students' mental health status during the COVID-19 pandemic and suggest measures to prevent depressive anxiety among them. METHOD: Undergraduate and graduate students at one national and two private universities in the Kansai region were surveyed. The Kessler Psychological Distress Scale-6 was used to assess the students' mental health. Questions were included to assess students' awareness of COVID-19 and changes in lifestyle habits, including drinking, smoking, gaming, and other addictive habits. The University of Tokyo Health Sociology's version of the Sense of Coherence Scale was used to assess the ability to cope with stressors. RESULTS: More than 50% of undergraduate and graduate students felt more than mild depressive anxiety and approximately 11% felt severe depressive anxiety, indicating that anxiety about the future worsened the levels of depressive anxiety. Life with reversed day and night schedules was associated with the worsening of depressive anxiety levels, but a high sense of coherence was associated with decreased levels of depressive anxiety. CONCLUSION: COVID-19 pandemic triggered isolation which led to worsening the mental health of undergraduate and graduate students. Psychological support for lifestyle and a sense of coherence is necessary to prevent mental health deterioration among isolated students. LIMITATIONS: As we were unable to contact all students, the sample bias may affect interpretation of the data.

Altered plasma levels of lysophospholipids in response to adrenalectomy of rats.

The aim of this study was to investigate effects of reduced stress hormone by adrenalectomy on rat plasma levels of lysophosphatidic acid (LPA) and other lysophospholipids. We measured activities of lysophospholipase D (lysoPLD) in plasma and lipid phosphate phosphatase (LPP) in blood by determining choline and inorganic phosphate, respectively. LPA, lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS) and lysophosphatodylglycerol were quantified by LC-MS/MS. In adrenalectomized rats, plasma levels of LPA, LPE, LPS and LPI, but not LPC, were increased. The increased level of LPA were due to decreased LPC level, increases plasma activity of lysoPLD toward LPC and decreased LPP activity toward LPA. Daily injections of deoxycoricosterone into rats selectively reversed increased level of LPS. Our results suggest enzymatic mechanism for increased plasma level of LPA, and indicate that the circulating levels of lysophospholipids including LPA in rats are differently affected by artificial suppression of release of adrenergic hormones.

Identification of human glycerophosphodiesterase 3 as an ecto phospholipase C that converts the G protein-coupled receptor 55 agonist lysophosphatidylinositol to bioactive monoacylglycerols in cultured mammalian cells.

A family of glycerol-based lysolipid mediators comprises lysophosphatidic acid as a representative phospholipidic member but also a monoacylglycerol as a non-phosphorus-containing member. These critical lysolipid mediators are known to be produced from different lysophospholipids by actions of lysophospholipases C and D in mammals. Some members of the glycerophosphodiesterase (GDE) family have attracted recent attention due to their phospholipid-metabolizing activity. In this study, we found selective depletion of lysophosphatidylinositol among lysophospholipids in the culture medium of COS-7 cells transfected with a vector containing glycerophosphodiester phosphodiesterase 2 (GDPD2, GDE3). Thin-layer chromatography and liquid chromatography-tandem mass spectrometry of lipids extracted from GDE3-transfected COS-7 cells exposed to fluorescent analogs of phosphatidylinositol (PI) revealed that GDE3 acted as an ecto-type lysophospholipase C preferring endogenous lysophosphatidylinositol and PI having a long-chain acyl and a short-chain acyl group rather than endogenous PI and its fluorescent analog having two long chain acyl groups. In MC3T3-E1 cells cultured with an osteogenic or mitogenic medium, mRNA expression of GDE3 was increased by culturing in 10% fetal bovine serum for several days, concomitant with increased activity of ecto-lysophospholipase C, converting arachidonoyl-lysophosphatidylinositol, a physiological agonist of G protein-coupled receptor 55, to arachidonoylglycerol, a physiological agonist of cannabinoid receptors 1 and 2. We suggest that GDE3 acts as an ecto-lysophospholipase C, by switching signaling from lysophosphatidylinositol to that from arachidonoylglycerol in an opposite direction in mouse bone remodeling.

Distinct contributions of two choline-producing enzymatic activities to lysophosphatidic acid production in human amniotic fluid from pregnant women in the second trimester and after parturition.

The purpose of this study was to clarify whether human amniotic fluid (AF) contains a significant level of bioactive lysophosphatidic acid (LPA) and, whether autotaxin (ATX) is involved in the production of LPA, if present. Using LC-MS/MS, we found a higher ratio of levels of LPA and its precursor lysophosphatidylcholine (LPC) in AF collected after parturition than that in AF collected at the middle stage of pregnancy. We detected significant choline-producing enzymatic activity toward an exogenous LPC in AF at the middle stage of pregnancy, about half of which was ascribable to ATX. In AF collected after parturition, the ATX-independent choline-producing activity of glycerophosphcholine phosphodiesterase coupled to lysophospholipase A activity was increased in relative to the lysophospholipase D activity of ATX. These results suggest that the increased LPA/LPC ratio in AF at the term of pregnancy was due to not only a moderate increase in the level of LPC, but also an unknown mechanism involving epithelial cells bathed with AF.

A low level of lysophosphatidic acid in human gingival crevicular fluid from patients with periodontitis due to high soluble lysophospholipase activity: Its potential protective role on alveolar bone loss by periodontitis.

We previously detected a submicromolar concentration of lysophosphatidic acid (LPA) in human saliva. Here, we compare LPA concentrations in human gingival crevicular fluid (GCF) from patients with periodontitis and healthy controls, and examine how the local LPA levels are regulated enzymatically. The concentrations of LPA and its precursor lysophospholipids in GCF was measured by liquid chromatography-tandem mass spectrometry. The LPA-producing and LPA-degrading enzymatic activities were measured by quantifying the liberated choline and free fatty acid, respectively. The concentration of LPA in GCF of periodontitis patients was lower than that of healthy controls, due to higher soluble lysophospholipase activity toward LPA. LPA was found to prevent survival of Sa3, a human gingival epithelium-derived tumor cell line, activate Sa3 through Ca2+ mobilization, and release interleukin 6 from Sa3 in vitro. Furthermore, local injection of LPA into the gingiva attenuated ligature-induced experimental alveolar bone loss induced by oral bacteria inoculation in a rat model of periodontitis in vivo. A high concentration of LPA in human GCF is necessary to maintain normal gingival epithelial integrity and function, protecting the progression of periodontitis.

Synthesis and In Vitro Assessment of pH-Sensitive Human Serum Albumin Conjugates of Pirarubicin.

In a previous study, we reported on the development of a synthetic polymer conjugate of pirarubicin (THP) that was formed via an acid-labile hydrazone bond between the polymer and the THP. However, the synthetic polymer itself was non-biodegradable, which could lead to unexpected adverse effects. Human serum albumin (HSA), which has a high biocompatibility and good biodegradability, is also a potent carrier for delivering antitumor drugs. The objective of this study was to develop pH-sensitive HSA conjugates of THP (HSA-THP), and investigate the release of THP and the cytotoxicity under acidic conditions in vitro for further clinical development. HSA-THP was synthesized by conjugating maleimide hydrazone derivatives of THP with poly-thiolated HSA using 2-iminothiolane, via a thiol-maleimide coupling reaction. We synthesized two types of HSA-THP that contained different amounts of THP (HSA-THP2 and HSA-THP4). Free THP was released from both of the HSA conjugates more rapidly at an acidic pH, and the rates of release for HSA-THP2 and HSA-THP4 were similar. Moreover, both HSA-THPs exhibited a higher cytotoxicity at acidic pH than at neutral pH, which is consistent with the effective liberation of free THP under acidic conditions. These findings suggest that these types of HSA-THPs are promising candidates for further development.

Addition of high load of lysophosphatidic acid to standard and high-fat chows causes no significant changes of its circulating and peripheral tissue levels but affects body weight and visceral fat mass of mice.

Oral administration of lysophosphatidic acid (LPA), a critical intercellular lipid mediator, exerts wound healing and antiulcer effects on gastrointestinal system. To evaluate effects of food-derived LPA on body homeostasis, we measured LPA levels by liquid chromatography-tandem mass spectrometry in chows, feces, plasma, liver, and visceral fat of mice fed a normal or high-fat chow supplemented with or without LPA-rich soybean phospholipids for 30 days. Reductions in daily body weight gains and visceral fat mass were mainly related to lower chow intake by mice fed the LPA-rich high-fat chow, whereas reduced body weight gains and fat mass were mainly related to decreased intestinal triacylglycerol absorption in mice fed LPA-rich chow. Our results showed no significant increase in plasma, liver, or adipose LPA levels, even if a quite high LPA concentration (2.0%) in chows was ingested daily, suggesting limited effects of food-derived LPA on the lumen side of the digestive tract. © 2018 BioFactors, 44(6):548-557, 2018.

Peripheral tissue levels and molecular species compositions of N-acyl-phosphatidylethanolamine and its metabolites in mice lacking N-acyl-phosphatidylethanolamine-specific phospholipase D.

N-acylethanolamines (NAEs), a class of lipid mediators, are produced from N-acyl-phosphatidylethanolamine (NAPE) by several pathways, including the direct release by NAPE-specific phospholipase D (NAPE-PLD) or the multistep pathway via sn-glycero-3-phospho-N-acylethanolamine (Gp-NAE). Using liquid chromatography-tandem mass spectrometry, we compared peripheral tissue levels of NAPE, Gp-NAE and NAE in NAPE-PLD-deficient (NAPE-PLD-/-) and wild type (WT) mice. NAPE-PLD was suggested to play a major role in the NAPE degradation in heart, kidney, and liver, but not in jejunum, because the NAPE levels except jejunum were significantly higher in NAPE-PLD-/- mice than in WT mice. The deletion of NAPE-PLD failed to alter the NAE levels of these tissues, suggesting its limited role in the NAE production. The enzyme assays with tissue homogenates confirmed the presence of NAPE-PLD-independent pathways in these peripheral tissues. Gp-NAE species having an acyl moiety with 22 carbons and 6 double bonds was enriched in these peripheral tissues. As for sn-2 acyl species of NAPE, 18:2-acyl-containing NAPE species were predominant over 18:1-containing species in heart, liver, and jejunum. Our results show that both molecular species composition of NAPE, NAE and Gp-NAE and their dependencies on Napepld are different among the peripheral tissues, suggesting that each tissue has distinct metabolic pathways and these NAE-containing lipids play tissue-specific roles.

Daily Intake of High-Fat Diet with Lysophosphatidic Acid-Rich Soybean Phospholipids Augments Colon Tumorigenesis in Kyoto Apc Delta Rats.

BACKGROUND: Oral administration of lysophosphatidic acid (LPA) was shown to attenuate gastric ulceration in rats and mice but aggravate intestinal tumorigenesis in mice. AIMS: The present study examined whether dietary LPA induces or prevents development of colorectal tumor in rats. METHODS: Kyoto Apc Delta rats fed high-fat diet with or without an LPA-rich soybean phospholipid mixture (LSP, 0.1 or 1%) were treated with azoxymethane and dextran sodium sulfate to induce colorectal tumorigenesis. Rats were killed 15 weeks after azoxymethane treatment, and size, total number, location, and severity of colorectal tumors were assessed. Expression of mRNA of LPA receptors in rat colon tissue was assayed. RESULTS: Rats fed the diet supplemented with 1% LSP had a higher number of tumors 2-4 mm long compared than those with or without 0.1% LSP. The mean distance of tumors >4 mm long from the anus was significantly higher than those of tumors <2 and 2-4 mm long in rats fed 1% LSP-supplemented diet. Supplementation of the diet with 0.1% LSP decreased mRNA expression of LPA5 in colon tumors of rats. CONCLUSIONS: Dietary supplementation of LPA-rich phospholipids dose-dependently augmented colorectal tumorigenesis. Decreased expression of LPA5 in colon tumors may be relevant to augmented tumorigenesis.

Reduced rat plasma lysophosphatidylglycerol or lysophosphatidic acid level as a biomarker of aristolochic acid-induced renal and adipose dysfunctions.

AIMS: Food products and diet pills containing aristolochic acid (AA) are responsible for a rapid progression of nephropathy associated with reduced body weight in human beings. In this study, we investigated the relationship of dietary NaCl and lysophospholipid (LPL) plasma levels to body weight gain in AA-treated rats. MAIN METHODS: Male rats receiving a salt-deficient chow, normal salt chow or high salt chow were injected intraperitoneally daily with AA for 15days. Body weight, visceral fat mass, food intake, levels of LPL in plasma and its synthesized enzyme were investigated. KEY FINDINGS: Body weight gain, visceral fat mass and daily food intake were smaller in AA-treated rats than those of control rats, regardless of dietary salt concentration. AA treatment decreased plasma levels of major lysophosphatidic acid (LPA) molecular species in rats fed the normal or high-salt chow but not the salt-deficient chow, whereas both the plasma lysophospholipase D activity and kidney mRNA level of autotaxin of AA-treated rats fed chow with defined salt concentrations were lower than those of control rats. Plasma levels of major molecular species of lysophosphatidylglycerol (LPG) in AA-treated rat groups fed chow with defined salt concentrations were lower than those of control rats. SIGNIFICANCE: Plasma levels of LPG and LPA seem to be relevant to the reduced body weight gain and fat mass due to AA treatment.

Reduced kidney levels of lysophosphatidic acids in rats after chronic administration of aristolochic acid: Its possible protective role in renal fibrosis.

Aristolochic acid (AA) is considered to be a causative agent for progressive interstitial renal fibrosis, leading to AA nephropathy. Lysophosphatidic acid (LPA) is a mediator in the onset of renal fibrosis. In this study, we analyzed the molecular species of LPA and its precursor lysophospholipids in kidney tissue from rats exposed to AA. Daily intraperitoneal injections of AA for 35 days to rats gave rise to fibrosis in kidney, decreased the kidney levels of LPA, lysophosphatidylserine and lysophosphatidylinositol. In rat renal cell lines (NRK52E and NRK49F), AA-induced cytotoxicity was potentiated by Ki16425, LPA1,3 receptor antagonist. The level of mRNA encording α-smooth muscle actin was significantly increased by AA-treatment only in NRK52E cells, while the mRNA level of collagen III was decreased in both NRK52E and NRK49F cells. These results suggest that endogenous LPA in rat kidney prevents AA-induced renal fibrosis.

The potential protective role of lysophospholipid mediators in nephrotoxicity induced by chronically exposed cadmium.

Cadmium is a hazardous metal whose chronic exposure induces renal failure due to fibrosis, but the mechanisms are not well known. In this study we analyzed the molecular species of lysophosphatidic acid (LPA) and related phospholipids, together with their metabolic enzyme activity, in plasma from Wistar rats exposed up to 300ppm Cd(2+) in drinking water for 114days. Exposure of 300ppm Cd(2+) for 114days enhanced autotoxin (ATX)/lysophospholipase D activity, but significantly lowered the total levels of LPA and lysophosphatidylethanolamine. Interestingly, the total level of sphingosine-1-phosphate (S1P) was elevated dose-dependently by Cd(2+). Cultured rat kidney-derived interstitial fibroblast cells, NRK49F cells and proximal epithelial cells, NRK52E cells, were both responsive to the protective action of LPA or S1P against Cd(2+) toxicity. The former cell expresses ATX RNA. In conclusion, the elevation of LPA-producing enzyme activity and S1P concentrations in plasma after exposure of rats to Cd(2+) would protect from the renal toxicity of Cd(2+).

Lysophospholipids and lysophospholipase D in rabbit aqueous humor following corneal injury.

We previously found that lysophosphatidic acid (LPA)-like activity eliciting Cl(-) currents in Xenopus oocytes is increased in rabbit aqueous humor (AH) following corneal freeze wounds. The purpose of this study was to examine whether actual levels of LPA in AH from wounded eyes are higher than those from control eyes, and to determine the sources and enzymatic pathways of AH LPA in control and wounded conditions. Lysophospholipase D (lysoPLD) activity was measured by the enzymatic determination of choline following incubation of AH samples with exogenous lysophosphatidylcholines (LPCs). The molecular species compositions of LPA and LPC in fresh and incubated AH were determined by liquid chromatography-tandem mass spectrometry. A high, but similar activity of lysoPLD in the samples from both control and freeze-wounded eyes was detected. Its enzymatic properties resemble those of plasma lysoPLD, identified as autotaxin. Levels of LPCs, predominant substrates of lysoPLD in AH, were several times higher in the AH samples from injured eyes than those from the control eyes. Our results suggest that lysoPLD is constitutively released from corneal tissues and/or ciliary body into the AH, with no injury-induced increase in release following freeze-wounding. They also suggest that wound-induced increases in LPA-like biological activity are due to linoleoyl species-rich molecular composition in AH from wounded eyes. A possible mechanism of the altered molecular composition is an increase in the AH concentrations of LPCs, linoleoyl species of which are preferentially converted to corresponding unsaturated LPA by the constitutively active lysoPLD.

Comparison of lysophospholipid levels in rat feces with those in a standard chow.

Although lysophospholipids have attracted much attention due to their diverse physiological activities through their specific receptors, little is known about their metabolic fates in mammalian digestive systems after their ingestion as a minor food component. In this study, we analyzed five lysophospholipids in lipid extracts of a standard rat chow and feces of rats fed the chow by two-dimensional thin layer chromatography and liquid chromatography-tandem mass spectrometry. The most abundant lysophospholipid in the rat chow was lysophosphatidylcholine followed by lysophosphatidylethanolamine, lysophosphatidic acid (LPA), lysophosphatidylinositol and lysophosphatidylserine (LPS) in an increasing order, but their concentrations were very low in rat feces. Among the molecular species of LPS in the chow, only saturated species were detected in the feces in significant amounts. In addition, several molecular species of LPA remained in the feces in variable portions (saturated > monounsaturated > polyunsaturated). These results suggest that a portion of ingested LPA and LPS reach the rat large intestine, affecting physiological colon functions.

Presence of bioactive lysophosphatidic acid in renal effluent of rats with unilateral ureteral obstruction.

AIMS: Abnormal production of lysophosphatidic acid (LPA), an important lysophospholipid mediator, in the kidney was examined to participate in the pathogenesis of renal fibrosis in rats. The secretory lysophospholipase D activity of autotoxin was considered as a possible pathway for extracellular production of LPA in the pathological renal fluids. MAIN METHODS: In this study of rats with unilateral ureteral obstruction for two weeks, we measured concentrations of LPA and its precursor, lysophosphatidylcholine stored in the urinary bladder and present in the swollen pelvis of the ligated kidney as well as the corresponding blood plasma by liquid chromatography-tandem mass spectrometry. KEY FINDINGS: We found that concentrations of LPA and lysophosphatidylcholine accumulated in the effluent in the swollen pelvis of the ligated kidney of unilateral ureteral obstruction rats were much higher than those in the urinary bladder. The molecular species composition of LPA in the former was considerably different from that in the blood plasma, indicating the involvement of an additional source other than the blood circulation supplying LPA to the effluent in the swollen kidney. A potential mechanism is increased release of LPA from activated renal cells in the ureter-ligated kidney. SIGNIFICANCE: Both pathways for supply of extracellular LPA may participate in the induction and progression of renal tubulofibrosis.